Methods of t cell production

Abstract

This invention relates to methods of producing a population of progenitor T cells comprising differentiating haematopoietic progenitor cells (HPCs) into progenitor T cells in the presence of a pyrimidoindole compound, such as methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b] indole-7-carboxylate (UM729) or (1R,4R)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine (UM171). The progenitor T cells may be matured, activated and expanded, for example for use in immunotherapy.

1 . A method of producing a population of progenitor T cells comprising;
 differentiating a population of haematopoietic progenitor cells (HPCs) into progenitor T cells in the presence of a pyrimidoindole compound.   
     
     
         2 . A method according to  claim 1  wherein the presence of the pyrimidoindole compound increases the proportion of HPCs that differentiate into progenitor T cells. 
     
     
         3 . A method according to  any one of the preceding claims  wherein the HPCs are differentiated by a method comprising culturing the population of HPCs in a lymphoid expansion medium supplemented with an effective amount of the pyrimidoindole compound. 
     
     
         4 . A method according to  claim 3  wherein the lymphoid expansion medium consists of a chemically defined nutrient medium supplemented with effective amounts of SCF, FLT3L, TPO, IL7 and the pyrimidoindole compound. 
     
     
         5 . A method according to  any one of the preceding claims  wherein the HPCs have a CD34+ phenotype. 
     
     
         6 . A method according to  any one of the preceding claims  wherein the HPCs are differentiated by a method comprising differentiating the HPCs into common lymphoid progenitor cells (CLPs) and differentiating the CLPs into progenitor T cells. 
     
     
         7 . A method according to  any one of the preceding claims  wherein the population of HPCs is produced in vitro from induced pluripotent stem cells (iPSCs). 
     
     
         8 . A method according to  claim 7  wherein the method comprises providing a population of iPSCs and differentiating the iPSCs into a population of HPCs. 
     
     
         9 . A method according to  any one of the preceding claims  wherein the progenitor T cells have a CD5+, CD7+ phenotype. 
     
     
         10 . A method for expanding progenitor T cells comprising;
 culturing the progenitor T cells in the presence of a pyrimidoindole compound.   
     
     
         11 . A method according to  claim 10  wherein the presence of the pyrimidoindole compound increases the proliferation of the progenitor T cells in the population. 
     
     
         12 . A method according to  claim 10 or claim 11  wherein expansion of the progenitor T cells is for at least 21 days 
     
     
         13 . A method according to  any one of the preceding claims  wherein the pyrimidoindole compound is a substituted pyrimido[4,5-b]indole. 
     
     
         14 . A method according to  claim 13  wherein the pyrimidoindole compound is methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b] indole-7-carboxylate. 
     
     
         15 . A method according to  claim 13  wherein the pyrimidoindole compound is (1R,4R)—N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine. 
     
     
         16 . A method according to  any one of the preceding claims , wherein the pyrimidoindole compound is present at a concentration less than 5 μM. 
     
     
         17 . A method according to any one of  claims 1 to 16  wherein the method further comprises introducing heterologous nucleic acid encoding an αβ TCR into the iPSCs, HPCs or progenitor T cells. 
     
     
         18 . A method according to  claim 17  wherein the heterologous nucleic acid encoding the αβ TCR is comprised in an expression vector. 
     
     
         19 . A method according to  claim 18  wherein the expression vector is a lentiviral vector. 
     
     
         20 . A method according to any one of  claims 17 to 19  wherein the αβ TCR is an affinity enhanced TCR. 
     
     
         21 . A method according to one of  claims 17 to 20  wherein the αβ TCR binds specifically to an MHC displaying a peptide fragment of a target antigen expressed by cells or specifically binds to a target antigen or peptide thereof expressed by cells independently of MHC presentation. 
     
     
         22 . A method according to  claim 21  wherein the αβ TCR binds specifically to an MHC displaying a peptide fragment of a tumour antigen expressed by the cancer cells or binds specifically to a tumour antigen or peptide fragment thereof expressed by cancer cells independently of MHC presentation. 
     
     
         23 . A method according to  any one of the preceding claims  comprising further differentiating the progenitor T cells to produce TCR αβ+ T cells. 
     
     
         24 . A method according to  claim 23  wherein the progenitor cells are further differentiated by a method comprising culturing the population of progenitor T cells in a T cell maturation medium. 
     
     
         25 . A method according to  claim 23 or claim 24  wherein the TCR αβ+ T cells have a CD8+CD4+ phenotype. 
     
     
         26 . A method according to any one of  claims 23 to 25  comprising activating and expanding the TCR αβ+ T cells to produce a population of T cells have a CD8+ single positive phenotype or a CD4+ single positive phenotype. 
     
     
         27 . A method according to any one of  claims 23 to 26  wherein the TCR αβ+ T cells specifically bind to cells expressing a target antigen. 
     
     
         28 . A method according to  claim 27  wherein the target antigen is a tumour antigen. 
     
     
         29 . A method according to  claim 28  wherein the TCR αβ+ T cells specifically bind to cancer cells expressing the tumour antigen. 
     
     
         30 . A method according to any one of  claims 23 to 29  further comprising isolating or purifying the TCR αβ+ T cells. 
     
     
         31 . A method according to  claim 30  wherein TCR αβ+ T cells are isolated by magnetic activated cell sorting. 
     
     
         32 . A method according to according to any one of  claims 23 to 31  further comprising concentrating the population of TCR αβ+ T cells. 
     
     
         33 . A method according to according to any one of  claims 23 to 32  comprising storing the population of TCR αβ+ T cells. 
     
     
         34 . A method according to any one of  claims 23 to 33  comprising formulating the population of TCR αβ+ T cells with a pharmaceutically acceptable excipient. 
     
     
         35 . A population of progenitor T cells produced by a method according to any one of  claims 1 to 22 . 
     
     
         36 . A population of TCR αβ+ T cells produced by a method according to any one of  claims 23 to 34 . 
     
     
         37 . A pharmaceutical composition comprising a population of TCR αβ+ T cells produced by a method according to any one of  claims 23 to 34  and a pharmaceutically acceptable excipient. 
     
     
         38 . A population of TCR αβ+ T cells produced by a method according to any one of  claims 23 to 34  for use in a method of treatment. 
     
     
         39 . A population of TCR αβ+ T cells produced by a method according to any one of  claims 23 to 34  for use in a method of treatment of cancer.